Lethal Acid Stress Research Articles

Effect οf οxygen availability and pH οn adaptive acid tolerance response of immobilized Listeria monocytogenes in structured growth media

The aim of the present study was to evaluate the effect of oxygen availability (aerobic, hypoxic and anoxic conditions) and sub-optimal pH (6.2 and 5.5) in a structured medium (10% w/V gelatin) on the growth of two immobilized L. monocytogenes strains (C5, 6179) at 10 °C and their subsequent acid resistance (pH 2.0, e.g., gastric acidity). Anaerobic conditions resulted in lower bacterial population (P < 0.05) (7.8–8.2 log CFU/mL) at the end of storage than aerobic and hypoxic environment (8.5–9.0 log CFU/mL), a phenomenon that was intensified at lower pH (5.5), where no significant growth was observed for anaerobically grown cultures. Prolonged habituation of L. monocytogenes (15 days) at both pH increased its acid tolerance resulting in max. 10 times higher t4D (appx. 60 min). The combined effect though of oxygen availability and suboptimal pH on L. monocytogenes acid resistance was found to vary with the strain. Anoxically grown cultures at pH 5.5 exhibited the lowest tolerance towards lethal acid stress, with countable survivors occurring only until 20 min of exposure at pH 2.0. Elucidating the role of oxygen limiting conditions, often encountered in structured foods, on acid resistance of L. monocytogenes, would assist in assessing the capacity of L. monocytogenes originated from different food-related niches to withstand gastric acidity and possibly initiate infection.

Influence of cold stress on the survival of Listeria monocytogenes Bug600 and ScottA in lethal alkali, acid and oxidative stress

The objective of this study was to determine the influence of cold stress (4 °C) on the survival of Listeria monocytogenes Bug600 and ScottA in lethal alkali, acid and oxidative stress in broth and in distilled water. Four distinct patterns were observed: (1) Survival of cold stressed L. monocytogenes was increased by 1–4 logs in lethal alkali stress (pH 12.3 NaOH or KOH); (2) Survival of cold stressed L. monocytogenes was decreased by 1–4 logs in lethal acid stress (pH 1.3 HCl or H3PO4); (3) Survival of cold stressed L. monocytogenes was increased by 1–4 logs in lethal oxidative stress (1500 ppm H2O2); and (4) No difference in survival of cold stressed L. monocytogenes was observed in lethal oxidative stress by chlorine (1000 ppm NaOCl) compared to control. These patterns were also consistently observed for survival of L. monocytogenes cells in lethal alkali, acid or oxidative stress after adaptation of cold stress at 4 °C in low nutrient conditions (1/10 TSBYE). These findings demonstrate that cold stressed cells of L. monocytogenes Bug600 and ScottA have greater tolerance to lethal alkali and some oxidative stresses that are commonly employed by the food industries to kill this foodborne pathogen.

DegP functions as a critical protease for bacterial acid resistance.

To counteract the lethal acid stress, bacteria explore such strategies as cytoplasmic decarboxylase-catalyzed proton consumption and periplasmic chaperone-assisted protein refolding. Here, we report a periplasmic protease-mediated acid resistance mechanism in Escherichiacoli. Deletion of the protease gene degP dramatically decreases the viability of late log or early stationary phase cells against the extreme acid stress (pH 2.3), which can only be minimally rescued by complementary expression of the protease-deficient DegP(S210A) mutant protein. Similarly, DegQ, a homolog of DegP, also contributes to the bacterial acid resistance, but SurA as an important periplasmic chaperone hardly exhibits protection effect. Invitro studies reveal that DegP completely loses its protease activity under acidic condition but is able to partially reactivate upon neutralization. Importantly, we demonstrate the interaction of DegP with typical cellular substrate proteins in cells during acid stress and/or recovery stages by using unnatural amino acid-mediated invivo photo-crosslinking, as well as the degradation of periplasmic proteins by DegP during recovery after acidic denaturation. These data illustrate the role of DegP in bacterial acid resistance conceivably via degrading those acid-induced misfolded proteins. Our findings, together with earlier reports, suggest a comprehensive acid resistance strategy adopted by bacteria such that in the ATP-deficient extra-cytoplasm, the inevitable misfolded proteins induced by acid stress are refolded by a chaperone (e.g., HdeA/HdeB) and/or cleaved by a protease (e.g., DegP/DegQ) while in the cytoplasm excessive protons are directly consumed or exported.

Vibrational spectroscopy combined with transcriptomic analysis for investigation of bacterial responses towards acid stress.

The ability of bacteria to tolerate acid stress plays an important role in their growth and survival. In particular, aciduric bacteria have several survival systems that prevent cell damage from acid stress. In this study, the effect of the bacterial stress induced by pre-adaptation at different pH values on the cellular macromolecules of Lactobacillus plantarum was investigated using Raman spectroscopy and Fourier transform infrared spectroscopy. The expression of key genes was also quantified to provide understanding of the transcriptional response of the cells to lethal acid stress conditions. Principal component analysis of the spectra exhibited marked differences in the spectral regions associated with carbohydrates, lipids, proteins, and nucleic acids for all acid-stressed cells compared to those of untreated control cells. The changes in spectroscopic and transcriptomic profiles that were observed revealed alterations in bacterial cell wall composition after acid treatment. The results suggest the existence of a complex bacterial stress response in which modifications of cellular compounds from pre-adaption at low pH are involved. This study demonstrates the potential application of vibrational spectroscopy techniques to discriminate between intact and injured bacterial cells as well as to study their stress responses after exposure to acid environments during food processing.

High-Salt Preadaptation of Vibrio parahaemolyticus Enhances Survival in Response to Lethal Environmental Stresses

Adaptation to changing environmental conditions is an important strategy for survival of foodborne bacterial pathogens. Vibrio parahaemolyticus is a gram-negative seafoodborne enteric pathogen found in the marine environment both free living and associated with oysters. This pathogen is a moderate halophile, with optimal growth at 3% NaCl. Among the several stresses imposed upon enteric bacteria, acid stress is perhaps one of the most important. V. parahaemolyticus has a lysine decarboxylase system responsible for decarboxylation of lysine to the basic product cadaverine, an important acid stress response system in bacteria. Preadaptation to mild acid conditions, i.e., the acid tolerance response, enhances survival under lethal acid conditions. Because of the variety of conditions encountered by V. parahaemolyticus in the marine environment and in oyster postharvest facilities, we examined the nature of the V. parahaemolyticus acid tolerance response under high-salinity conditions. Short preadaptation to a 6% salt concentration increased survival of the wild-type strain but not that of a cadA mutant under lethal acid conditions. However, prolonged exposure to high salinity (16 h) increased survival of both the wild-type and the cadA mutant strains. This phenotype was not dependent on the stress response sigma factor RpoS. Although this preadaptation response is much more pronounced in V. parahaemolyticus, this characteristic is not limited to this species. Both Vibrio cholerae and Vibrio vulnificus also survive better under lethal acid stress conditions when preadapted to high-salinity conditions. High salt both protected the organism against acid stress and increased survival under -20°C cold stress conditions. High-salt adaptation of V. parahaemolyticus strains significantly increases survival under environmental stresses that would otherwise be lethal to these bacteria.

Heat stress adaptation induces cross-protection against lethal acid stress conditions in Arcobacter butzleri but not in Campylobacter jejuni

The ability of many bacteria to adapt to stressful conditions may later protect them against the same type of stress (specific adaptive response) or different types of stresses (multiple adaptive response, also termed cross-protection). Arcobacter butzleri and Campylobacter jejuni are close phylogenetic relatives that occur in many foods of animal origin and have been linked with human illness (mainly diarrhoea). In the present study, sublethal stress adaptation temperatures (48 °C and 10 °C) and mild and lethal acid conditions (pH 5.0 and pH 4.0) were determined for A. butzleri and C. jejuni. In addition, it was evaluated whether these sublethal stress adaptations cause specific adaptive responses or cross-protection against subsequent mild or lethal acid stresses in these bacteria. The studies were conducted in broth adjusted to the different conditions and the results were determined by the dilution series plating method. It was shown that heat stress adapted A. butzleri (incubated for 2 h at 48 °C) were significantly more resistant to subsequent lethal acid stress (pH 4.0) than non-adapted cells at the 1 h time-point (p < 0.01 in Wilcoxon rank sum test). No specific adaptive responses against the stresses in A. butzleri or C. jejuni and no cross-protection in C. jejuni were found. The ability of heat stressed A. butzleri to tolerate later lethal acid conditions should be taken into account when designing new food decontamination and processing strategies.

Expression of GroEL and DnaK Proteins during the Acquisition of a Transitory Resistance to Lethal Stresses byActinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans is a periodontopathogenic bacterium associated with active lesions of localized aggressive periodontitis. Cells of A. actinomycetemcomitans grown to mid-log phase, diluted or undiluted, and subjected to a sublethal heat stress demonstrated a partial protection to a subsequent lethal heat stress. On the contrary, stationary phase heat-stressed cells did not exhibit this transient thermal protection. Heat-stressed cells at sublethal temperature showed a higher expression of GroEL and DnaK proteins by immunoblotting, which was in agreement with de novo synthesis. A. actinomycetemcomitans was found to be more sensitive to a pH than to a heat stress. A transient protection to a lethal acid stress was demonstrated when cells were previously subjected to a sublethal acid stress. On the contrary, such transient protection did not occur for alkaline stresses. DnaK but not GroEL proteins made up a high proportion of the total protein synthesized following acid stress. The protection phenomenon is highly complex and heat shock proteins, which may help A. actinomycetemcomitans to support subsequent lethal conditions, are not expressed in the same way during the different stresses investigated. Key words: Actinobacillus actinomycetemcomitans , periodontitis, heat shock protein, stress tolerance.

The acid stress response of the cyanobacterium Synechocystissp. strain PCC 6308.

The cyanobacterium Synechocystissp. strain PCC 6308 has been shown to exhibit predictable physiological responses to acid stress. Originally isolated from a Wisconsin lake, this cyanobacterium grows optimally under alkaline conditions in the laboratory. After acid stress at a pH of between 4.4 and 7.7, cells return to exponential growth following a lag phase. The organism's response to this tolerable acid stress involves cell concentration-dependent neutralization of the external medium to pH 6 or above within 5 min, maintenance of a transmembrane pH gradient, and maintenance of photosystem II efficiency. Lethal acid stress, at a pH below 4.4, results in the formation of aggregates of denatured proteins observed as granules near the cell periphery, the disruption of the transmembrane pH gradient, cell color change to blue, and damage to photosystem II.

Role of sigma(B) in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes.

To determine the contribution of sigma B (sigma(B)) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50 degrees C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45 degrees C, 5% ethanol, or pH 4.5). The DeltasigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the DeltasigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the DeltasigB strain. These results suggest the existence in L. monocytogenes of both a sigma(B)-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. sigma(B) contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The DeltasigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the DeltasigB strain lost viability more rapidly than the parent strain. sigma(B) contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that sigma(B) plays a role in protecting L. monocytogenes against environmental adversities.

Identification de protéines de stress chez Lactobacillus delbrueckii bulgaricus par électrophorèse bidimensionnelle

Identification of stress proteins in Lactobacillus delbrueckii bulgaricus by two-dimensional electrophoresis. We studied Lactobacillus bulgaricusadaptation to an acid stress. In L. bulgaricus, adapted cells (30 min incubation at pH 4.75) were ~250 fold more tolerant to lethal acid stress (30 min at pH 3.6) than non adapted ones. In order to identify proteins induced during the adaptation, de novo protein synthesis was monitored by 35 S methionine labeling of exponentially gro- wing cultures, under standard (pH 6) and adaptive (pH 4.75) conditions. After 2 dimensional (2-D) electrophoresis separation, the protein patterns were compared. At least 30 protein spots showed an increased radioactivity level under acidic conditions. We determined the N-terminal amino acid sequence of 3 highly induced proteins. By comparison to databases, they were identified as the heat shock proteins GroES, GroEL and DnaK. A differential induction was observed for GroES and GroEL, suggesting a post-transcriptionnal regulation mechanism. In this report we established a method for the study of acid stress response in L. bulgaricus. This method will also be useful for fur- ther studies on the adaptation of this bacterium to environmental variations. Lactobacillus bulgaricus / two dimensional electrophoresis / acid stress / adaptation / heat shock protein

Intracellular pH is a major factor in the induction of tolerance to acid and other stresses in Lactococcus lactis.

This study demonstrates that exposure of log-phase Lactococcus lactis subsp. cremoris 712 cells to mildly acid conditions induces resistance to normally lethal intensities of environmental stresses such as acid, heat, NaCl, H2O2, and ethanol. The intracellular pH (pHi) played a major role in the induction of this multistress resistance response. The pHi was dependent on the extracellular pH (pHo) and on the specific acid used to reduce the pHo. When resuspended in fresh medium, cells were able to maintain a pH gradient even at pHo values that resulted in cell death. Induction of an acid tolerance response (ATR) coincided with an increase in the ability of cells to resist change to an unfavorable pHi; nevertheless, a more favorable pHi was not the sole reason for the increased survival at acid pHo. Cells with an induced ATR survived exposure to a lethal pHo much better than did uninduced cells with a pHi identical to that of the induced cells. Survival following lethal acid shock was dependent on the pHi during induction of the ATR, and the highest survival was observed following induction at a pHi of 5.9, which was the lowest pHi at which growth occurred. Increased acid tolerance and the ability to maintain a higher pHi during lethal acid stress were not acquired if protein synthesis was inhibited by chloramphenicol during adaptation.

Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells.

The response of H+-ATPase to lethal acid stress is unknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cycles of lethal acid stress. Cells were grown to confluence on coverslips, loaded with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, and monitored for intracellular pH (pHi) recovery from an acid load. The rate of Na+-independent pHi recovery from an acid load in mutant cells was approximately fourfold higher than in parent cells (P < 0.001). The Na+-independent H+ extrusion was ATP dependent and K+ independent and was completely inhibited in the presence of diethylstilbestrol, N,N'-dicyclohexylcarbodiimide, or N-ethylmaleimide. These results indicate that the Na+-independent H+ extrusion in cultured medullary cells is mediated via H+-ATPase and is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H+-ATPase remained unchanged in mutant cells compared with parent cells. We propose that lethal acid stress results in increased H+-ATPase activity in inner medullary collecting duct cells. Upregulation of H+-ATPase could play a protective role against cell death in severe intracellular acidosis.

The effect of lethal acid stress on Na +/H + exchanger isoforms in cultured inner medullary collecting duct cells: deletion of NHE-2 and over expression of NHE-1

Cultured inner medullary collecting duct (mIMCD-3) cells express Na +/H + exchanger isoforms NHE-2 and NHE-1 (Soleimani et al. (1994) J. Biol. Chem. 269, 27973–27978). In the present studies we examined the effect of lethal acid stress on Na +/H + exchanger activity and isoform expression in mIMCD-3 cells. mIMCD-3 cells were incubated for 10 min with 20 mM ammonium, and exposed to an ammonium-free acidic solution (pH 6.0) for 120 min. Thereafter, cells were recovered and grown in normal culture media. The surviving clones were isolated and subjected to two additional cycles of acid stress. A mutant clone was isolated and characterized for Na +/H + exchange activity and isoform expression. The mutant mIMCD-3 clone demonstrated significant over-expression of Na +/H + exchange activity as assessed by acid-stimulated 22Na influx (11.56 nmol/mg protein in mutant vs. 4.06 nmol/mg in parent cells, P < 0.001, n = 4) and sodium-dependent pH i recovery from an acid load (0.55 pH/min in mutant vs. 0.28 pH/min in parent cells, P < 0.01, n = 6). A dose-response inhibition of the exchanger showed that the mutant cells were very sensitive to dimethylamiloride (IC 50 158 nM in mutant vs. 889 nM in parent mIMCD-3 cells, P < 0.001). To compare the Na +/H + exchanger isoforms in mutant and parent mIMCD-3 cells, poly(A) + RNA was isolated from each group and probed with radiolabeled NHE-1 or NHE-2 cDNA. The expression of NHE-1 mRNA was increased by ∼100% in mutant cells. The NHE-2 mRNA, on the other hand, was found to be absent in mutant mIMCD-3 cells. Examination of the regulatory mechanisms of the Na +/H + exchanger isoforms in parent mIMCD-3 cells, which express NHE-2 and NHE-1, and mutant mIMCD-3 cells, which only express NHE-1, would be helpful in elucidating the roles of NHE-2 and NHE-1 in inner medullary collecting duct cells.

Survival, Blood Osmolality, and Gill Morphology of Juvenile Yellow Perch, Rock Bass, Black Crappie, and Largemouth Bass Exposed to Acidified Soft Water

When exposed to a range of pH from 7.0 to 4.0 in soft water (1 mg Ca2+/L), juvenile rock bass Ambloplites rupestris, black crappie Pomoxis nigromaculatus, and largemouth bass Micropterus salmoides showed a capacity to osmoregulate and survive for up to 30 d at pH 4.5 and above. Juvenile yellow perch Perca flavescens maintained osmoregulatory control through 58 d at pH 5.0. All four species lost osmoregulatory control at pH 4.0, and death of fish ensued within a few days after blood osmolality declined to about 200 mosmol/kg or less (normal values, about 300 mosmol/kg). After 58 d of exposure to pH 4.0, mean blood osmolality of yellow perch was 218 mosmol/kg, and these fish were severely emaciated and moribund. Rock bass, black crappie, and largemouth bass all died by days 29, 16, and 9, respectively, when exposed to pH 4.0. However, when exposure to pH 4.0 was halted and pH was raised to 7.0 before death, blood osmolality returned to near-initial values within 15 d. Cessation of feeding also was noted at pH 4.0. Examination of gills showed progressively increased pathology with longer exposures to lower than normal pH. Among fish exposed to low pH, gill hyperplasia was present most often, but epithelial hypertrophy, chloride-cell proliferation, chloride-cell degeneration, edema, and vacuolization of the tissues also were observed. Morphological changes that were observed in the three centrarchids at pH values above pH 4.0 suggested that gill pathology may be a more sensitive indicator of potentially lethal acid stress than blood osmolality.